Calcium switch. Does the calcium switch of cardiac troponin resemble that of skeletal troponin in so far that it also requires the binding of 4 calcium ions in order for the actin filament to be turned on? How many different states can be adopted by the regulated actin filament? Whereas our work in combination with X-ray data suggests the existence of at least 3 states of activity it does not exclude the possibility that additional intermediate states can be adopted. Therefore we intend to study by a variety of methods how the potentiated state obtained in the presence of tropomyosin alone is modified by the addition of troponin and of troponin subunits such as TN-T and TN-I. Similarly we intend to compare the off state maintained by calcium-free troponin to the off state maintained by TN-I. Mechanism of switching over from one state to the other. A test of our hypotheses is provided by a closer study of the mechanism by which NEM-treated myosin can switch on a regulated actin filament in the absence of calcium and under conditions where the concentration of rigor complexes should be very low. A better understanding of the mechanism of switching may come from studies that compare switching off by TN-I in the absence of tropomyosin and in its presence. The effect of phosphorylation of actin or TN-T by protein kinase on the cooperative interactions within the regulated filament will be studied as well as such effects of phosphorylation of TN-I by phosphorylase kinase.